Recent advances have allowed for the generation of transgenic avians that express heterologous proteins in their oviduct cells. The avian oviduct contains an infundibulum, magnum, isthmus, shell gland, vagina and cloaca (Etches, Reproduction in Poultry. 1996, New York, N.Y.: CABI Publishing. 318). An ovulated ovum enters the tract through the infundibulum and continues to the magnum where the majority of egg white proteins, such as ovalbumin, ovomucoid and lysozyme, are produced by tubular gland cells and deposited on the ova. (Palmiter, J Biol Chem, 1972. 247: 6450-61; Yu and Marquardt, Biol Reprod, 1973. 8:283-98). Expression of the major egg white proteins is controlled by hormone responsive elements of the respective promoters (Palmiter, J Biol Chem, 1972. 247:6450-61; Schimke et al., Basic Life Sci, 1973. 1:123-35; Mulvihill and Palmiter, J Biol Chem, 1977. 252: 2060-8; McKnight and Palmiter, J Biol Chem, 1979. 254:9050-8; Mulvihill and Palmiter, J Biol Chem, 1980. 255:2085-91; Shepherd et al., J Cell Biol, 1980. 87: 142-51; Palmiter et al., J Biol Chem, 1981. 256: 7910-6; Sanders and McKnight, Endocrinology, 1985. 116: 398-405).
Chicken oviduct cells, when stimulated by steroid hormones during egg-laying, secrete three principal polypeptides, ovalbumin, ovomucoid and lysozyme (Tsai et al., (1978) Biochemistry 17: 5773-5779). The mRNA transcript encoding ovalbumin constitutes about 50% of the total mRNA of these cells. Ovomucoid and lysozyme mRNAs contribute about 6.6% and 3.4% respectively of the total mRNA of the steroid stimulated cells. (Hynes et al. (1977) pp 932). The ability of the avian oviduct to express large amounts of various proteins makes it an attractive target for producing heterologous proteins. To date, the most common method of expressing heterologous proteins in avian oviducts is through the production of transgenic hens. In one example, transgenic hens expressing human Interferon alpha-2b (hIFN α2b) were generated (Rapp et al., Transgenic Research, 2003). Also, transgenic hens were generated that express human monoclonal antibody in their egg white. U.S. Pat. No. 6,730,822, issued May 5, 2004, and U.S. patent application Ser. No. 10/463,980, filed Jun. 17, 2003 filed internationally as PCT/US04/01833, U.S. patent application Ser. No. 09/877,374, filed Jun. 8, 2001 filed internationally as PCT/US02/02454, U.S. patent application Ser. No. 10/679,034, filed Oct. 2, 2003 filed internationally as PCT/US02/29878, and U.S. patent application 10/856,218, filed May 28, 2004 filed internationally as PCT/US04/16827 disclose methods and compositions useful for the generation of transgenic avians for production of heterologous proteins. The disclosure of this issued US patent and these four pending patent applications and their corresponding PCT applications are incorporated herein by reference.
Primary cultures of oviduct tissue can be generated by removing the magnum section of a sexually mature hen treated with estrogen (Sanders and McKnight, Endocrinology, 1985. 116: 398-405). The tissue is digested with collagenase and dispase to liberate small cell clumps that are cultured for short durations. Cells collected and cultured in this manner typically die or differentiate into cells that do not produce egg white protein within three days making primary cultures of normal oviduct cells unsuitable for use in large scale in vitro production of heterologous proteins. Accordingly, improved methods for producing heterologous proteins in avian oviduct cells are needed.